The nature and sequence of cell-derived and environmental signals for progressive development of the "fully activated" macrophage (M0) have not been systemmatically examined. The endotoxin (LPS) "hyporesponsive" C3H/HeJ mouse strain provides a unique model for analyzing differentiation stimuli since these mice have genetic defects in M0 activation and M0 sensitivity to LPS. We will utilize C3H/HeJ M0 cultures to "map" the order of the differentiative signals by examining the responses of these cells to purified lymphokine and cytokine preparations in combination with each other and with LPS. We will prepare three classes of purified differentiative factors, including Gamma-interferon (Gamma-IFN), colony stimulating factor (CSF), and, if possible, a preparation of macrophage activating factor (MAF) free of Gamma-IFN and CSF activities. Our proposed purification procedures are based on extensive preliminary data using standard biochemical techniques. We will also prepare a monoclonal anti-Gamma-IFN reagent to be used as a specific blocking agent and as an additional tool for purifying Gamma-IFN. We will define the order of signals required for M0 activation by examining the sequence of development of specific M0 cell surface markers (Fc receptor, Ia antigen, and the "M0 activation" antigen) and by correlating the expression of these surface markers with the acquisition of new functional capabilities, including enhanced Fc and C3b-mediated phagocytosis, enhancement of M0 LPS sensitivity, and the ability to kill tumor cells in vitro. The identification of the specific sequences of intra- and intercellular signals that result in the development of the fully activated M0 could provide novel therapeutic approaches for diseases in which activated M0s have beneficial or harmful roles. The ability to stimulate activation of host M0s might benefit patients with neoplasms or infections with intracellular pathogens, whereas the ability to prevent M0 activation and resulting toxic effects might benefit patients with chronic inflammatory diseases such as rheumatoid arthritis or periodontal disease. An improved understanding of the relationships between the state of M0 activation and LPS sensitivity might provide insights for successful treatment of septic shock or other complications of gram negative bacillary infections.